PROJECT SUMMARY Prenatal or neonatal exposure to drugs of abuse such as cocaine and ethanol has been shown to disrupt neurogenesis and/or gliogenesis in the developing cerebral cortex, and induce functional abnormalities late in life. Proper formation of the cortex depends on the orderly production of a large number of diverse neurons, as well as glial cells. Radial glial cells have been shown to be a predominant population of neural progenitor cells in the developing cortex. In addition to their well-characterized role in supporting neuronal migration, radial glial progenitors (RGPs) actively divide to proliferate and to generate neurons and glial cells either directly or indirectly. The division mode and dynamics of RGPs essentially determine the number and types of neurons and glia in the cortex; however, the precise behavior and lineage progression of RGPs and the underlying molecular regulation are poorly understood. The long-term goal of this project is to systematically delineate RGP behavior and progeny output at the cellular and molecular levels. RGPs are neither synchronized in division dynamics nor homogenous in division pattern and progeny output. This calls for a systematic and quantitative analysis of the precise mitotic behavior and progeny output of RGPs in vivo at the single cell resolution. Recently, we exploited the unprecedented resolution of mosaic analysis with double markers (MADM) on progenitor division and progeny output, and performed a systematic and quantitative clonal analysis of RGP division and lineage progression. We revealed, for the first time, that RGPs progress through a remarkably deterministic and orderly program in proliferation, neurogenesis, and gliogenesis. Based on strong published and preliminary data, the central hypothesis of this application is that the behavior and output of individual RGPs are highly programmed at the cellular and molecular levels to produce a correct number and type of neurons and glia in the cortex. This hypothesis will be tested by 1) systematically and quantitatively examine the number, type, and organization of astrocytes and/or oligodendrocytes generated by individual RGPs at different embryonic stages using MADM, and 2) elucidate the molecular programs that regulate RGP lineage progression in proliferation, neurogenesis, and gliogenesis by performing in-depth real time single-cell transcriptome analysis of RGPs across different embryonic stages using CEL-seq in conjunction with loss-of- function studies using mouse genetics and/or CRISPR/CAS9 approaches. By integrating a battery of cutting- edge techniques, the proposed research will provide fundamental new molecular and cellular insights into RGP behavior and cortical neurogenesis and gliogenesis. This contribution will be significant because it will not only advance the basic knowledge of cortical histogenesis, but will also expand our understanding of the underlying cause of drugs of abuse-induced brain damage or other devastating developmental brain disorders with cortical abnormalities such as microcephaly, macrocephaly, and autism, and thereby potentially identify important molecular and cellular targets for diagnosis and treatment.